T cell subset profile in healthy Zambian adults at the University Teaching Hospital

Introduction Symptom-free human immunodeficiency virus antibody-negative Zambian adults (51 subjects, aged 20 to 62 years, 33.3% women and 66.7% men) were studied to establish T cell subset reference ranges. Methods We carried out across sectional study at the University Teaching Hospital, Lusaka. Blood samples were collected from healthy donor volunteers from hospital health care staff, between February and March 2015. Immunopheno typing was undertaken to characterize Tcell subsets using the markers CD3, CD4, CD8, α4β7, Ki67, CD25, CCR7, CD54RA, CD57, CD28, CD27 and HLA-DR. Results Among 51 volunteers, Women had significantly higher absolute CD4 count (median 1042; IQR 864, 1270) than in men (671; 545, 899) (p=0.003). Women also had more CD4 cells expressing homing, naïve, effector and effector memory T cell subsets compared to men. However, in the CD8 population, only the effector cells were significantly different with women expressing more than the males. Conclusion We provide early reference range for T cell subsets in Zambian adults and conclude that among the African women some T cell subsets are higher than men.


Introduction
Many AIDS-related research studies are being conducted in Zambia and other African nations with a high prevalence of HIV infection.
Common to these studies, is the measurements of total CD4 cell count, CD4 percentage and CD4/CD8 ratio in the peripheral blood.
The enumeration of circulating CD4 and CD8 lymphocyte subsets is important because these cells are perturbed during HIV infection.
Additionally, CD4 and CD8 measurements have been found to be useful surrogates for determining the risk of progression of HIV infection and are widely used in observational studies and AIDS clinical trials [1]. In low-income settings, HIV/AIDS related deaths are higher than in high-income settings [2]. One of the causes for this increased dearly mortality is having very low CD4 count at the start of antiretroviral therapy (ART) [3]. Studies conducted in Zambia have revealed that T cell subsets may be an accurate predictor of early mortality in HIV-infected adults [4] as well as in children [5] starting ART. However, there are no comparative data especially on normal parameters for T cell subsets in healthy adults.
Several studies have evaluated CD4 and CD8 lymphocyte subsets in children [6][7][8][9] and adults in Western nations [10][11][12][13][14][15] and other countries [16][17][18][19]. However, data on normal ranges of CD4 and CD8 T cell subsets in Zambia are generally lacking. Thus, because of the lack of normal reference ranges for CD4 and CD8 T cell subset parameters in healthy African subjects, many investigators interpret their data using values that have been derived from populations in Europe and the United States. It would therefore be helpful to establish appropriate normal reference values for T cell subsets especially in African populations. In this study, we describe CD4 and CD8T cell subset reference ranges obtained by studying 51 symptom-free HIV-seronegative Zambians.  Antibodies used were conjugated to the following fluorochromes as follows: CD45-PerCP CD4-PE CD3-FITC.

Characteristics of study participants
There were more men than women among the staff members recruited to this study, and the majority of these were in formal employment with a median age of 29 (IQR, 25-37) years (Table 1).
Haemoglobin, body mass index (BMI) and grip strength results for both male and female participants were within international normal ranges. As expected, grip strength differed substantially by sex.

CD4 + and CD8 + T cell subset distribution in healthy adult volunteers
Non-viable (CD4 + 7AAD + ) CD4+ T cells were completely absent in the healthy participants (data not shown). Figure 1 shows that majority of the CD4+ T cells in healthy adults were naïve

Discussion
In order to generate a reference group, a six colour FACSVerse instrument was used to characterise the CD4 + and CD8 + T cells in HIV-negative adults in a Zambian population. Overall, we found more CD8 cells than CD4 cells (ratio of 1.67) in our Zambian population, contrary to what has been reported in other African studies and in Europe [20,21]. With men and women taken together, the mean (SD) CD4 + cell count was 828 (336) cells/μL, which is higher than the values reported for healthy adult Ethiopians [22]. The values were comparable to those in Tanzanians [23] and lower than those reported for Ugandans [24].
Our data show that in healthy adults the majority of the CD4 + T cells were naïve. This finding was similar to findings from a study conducted in a similar cohort in San Francisco USA [25]. However, results for the healthy Ethiopians and Dutch showed lower numbers of naïve CD4 + T cells [26] compared to what was found in the Page number not for citation purposes 4 Zambian cohort. When naïve CD4 + T cells are stimulated by their cognate antigen presented by competent antigen-presenting cells they differentiate into specialized effect or and/or memory cells.

Effect or T cells induce cytolysis of infected cells or activate other lymphocytes and immune effect or cells, whereas memory T cells
maintain the capacity to respond rapidly to previously encountered antigens [27]. In the healthy volunteers, however, there were very few circulating effector and or memory CD4 + T cells in the blood compared to effector and effector memory CD8 + T cells.
Proliferating, proliferating and activated, activated and senescent CD4 + T cells in the healthy volunteers were very few. Since activation is triggered by the presence of antigen and is followed by proliferation, having few proliferating and activated cells may indicate absence of infectious disease and good T cell activation regulation in our healthy volunteers [28]. We also found a high number of CD4 + T cells expressing gut homing markers (about 35%) and activated gut homing (23%) in these healthy adults. In the CD8+population, approximately 39% were expressing gut homing cells. Gut homing cells are cells that are programmed to migrate to gut associated lymphoid tissues especially when there is an infectious challenge to the gut [32].
Finally, there were significant differences in absolute CD4 count between females and males in some of the subsets. The reference range of CD4 count was significantly higher in females than in the males, which is in agreement with other studies in Africa [33, 21].
We do not know whether this difference is due to environmental or genetic factors.
Our study has several limitations. All subjects were bled only once; therefore, it is possible that some subjects were in the "window" period of HIV infection, having been recently infected with HIV but not yet having developed anti-HIV antibodies. We did not perform medical or other laboratory examinations to evaluate the subjects´ general state of health but relied on self-reported symptom histories which may be inaccurate in excluding disease in individuals presenting for voluntary HIV screening. Therefore, the parameters used to describe the healthy volunteers were not exhaustive and as such, there could be other chronic viral infections such as cytomegalovirus or Epstein-Barr virus, which could have affected the CD4 and CD8 results. CD4 counts have been found to have significant diurnal and day-to-day variation in the same subjects and to vary with storage time and temperature [34]. While all blood samples were obtained during the late morning, stored at room temperature, and processed within a maximum of 2h after phlebotomy to minimize variability due to time of day and storage time and temperature among different subjects, we performed only single determinations of absolute CD4 count, and absolute CD8 count for each subject and cannot, therefore, comment on variability due to day-to-day variation among individuals.
Furthermore, the sample size was small and samples were drawn from the same area.

Conclusion
We propose that these reference intervals may be a useful point of comparison for studies on the immune system in Africa.
What is known about this topic

Acknowledgments
We would like to thank Kanekwa Betty Zyambo for her excellent technical support.